Clathrin-mediated endocytosis (CME) is a major route by which cells take up many nutrients and regulatory molecules and down-regulate membrane proteins in response to their ligands and environmental signals. Further importance of this pathway is exemplified by a number of human diseases that are associated with CME, including cancer, cardiovascular and neurological disorders, and entry of infectious viruses into cells. While many factors involved in CME have been identified and a large number of protein- protein or protein-lipid interactions for them have been described, assigning specific roles to these proteins remains a challenge to the field. There is still much to learn about how cargo are collected, the importance of the elaborate network of interactions and scaffolding functions of the endocytic machinery, how force is generated to invaginate and pinch off a vesicle, and how the timing and regulation of assembly and disassembly of these components coordinates this dynamic process. Moreover, aside from phosphoinositides, little is known about lipid requirements for formation of an endocytic vesicle. Studies in this lab have combined live cell imaging analysis with biochemistry, cell biology and the powerful molecular genetics methods of S. cerevisiae to study the endocytic process. In yeast, as in all eukaryotes, both clathrin and the actin cytoskeleton are critical for endocytosis. Furthermore, most endocytic factors identified in yeast have conserved counterparts in animal cells that are involved in CME. Work from several labs, including ours, has clearly validated yeast as an important model for discovering how this large molecular machine drives the internalization process. In this project we continue our efforts to dissect the roles of components of the endocytic machinery using the yeast system. We focus primarily on the novel F-BAR family of FCHO1/2 proteins (Syp1 in yeast), the role of clathrin light chain and regulation of its binding partner Sla2 (related to Hip1/R proteins in animl cells), and the roles of lipids in generating an endocytic vesicle. These studies will provide new insight into the mechanisms of endocytosis and vesicle formation during membrane transport in general.